PROJECT SUMMARY (See instructions); Toll-like receptor (TLR) and NOD-like receptor (NLR) signaling during the innate response to infection influences the host cell's permissiveness for cytosolic replication of bacteria and the development of protective immunity. However, it is not entirely clear how these signaling events initiated by engagement of TLRs and NLRs influences the capacity to confer protection against lethal bacterial infection through vaccination. It also is unclear how antibody bound to Francisella tularensis (Ft) in the form of an inactivated Ff-monoclonal antibody (IFf-mAb) complex, the vaccinogen (1,2) under evaluation in this proposal, alters innate signaling via TLR2 or NLRs and thereby the adaptive/protective immune response. Changes in a pathogen's capacity to resist redox-mediated host defenses or alter redox-based host cell signaling, as has been shown for the live vaccine strain (LVS) of Ft deficient for superoxide dismutase activity (3, 4), impacts both TLR and NLR-dependent inflammatory responses. Thus, pathogen-derived antioxidants likely influence the ability of different vaccinogens to engender adaptive/protective immunity. Therefore, we hypothesize that signals transduced via TLR2 and/or NLRs influence the efficacy of vaccination and these signals are impacted by antibody opsonization of Ft and/or the bacterium's proinflammatory capacity. This hypothesis will be tested by 1) Establishing the impact of mAb bound Ft and Ft antioxidant mutants on synergistic TLR2/NLR signaling during the innate immune (inductive) phase of the anti-Ft immune response at the molecular level and 2) Evaluating whether TLR2 and/or NLR signaling impacts induction of protective immunity after vaccination with various Ff formulations. Synergy: Results obtained in Subproject 2 will provide a new view of TLR and NLR-directed innate responses in the context of the ongoing vaccine strategies employed in Subproject 1, enabling refinement of these approaches. The signaling pathways involved in macrophage ROS/RNS production intersect those activated by FcR, TLR, and NLR stimulation, namely the PI3 kinase, NF-KB, and MAPK pathways; accordingly, the results of these Aims are significantly relevant to, and will help inform, experiments in Subproject 3. Further, this synergy will be facilitated by the provision of characterized antioxidant mutants from Subproject 3, thus enabling our Subproject to shed light on how Ft antioxidant pathways impact